Cervical cancer is the second-leading cause of cancer-related deaths among women worldwide, and epidemiologic and laboratory studies have shown that the human papillomavirus (HPV) E6 and E7 proteins of the "high-risk" subgroup of sexually transmitted HPVs play a causative role in over 90% of these cancers. The biochemical activity of the E6 proteins most clearly related to carcinogenesis is their ability to stimulate the ubiquitin-mediated degradation of the p53 tumor suppressor protein. This is dependent on the cellular ubiquitin ligase, E6AP. Biochemical analyses indicate that E6 functions to redirect or reprogram the substrate specificity of E6AP so that it ubiquitinates p53, a protein that it does not normally recognize. While many lines of evidence indicate that targeted degradation of p53 is critical to the role of HPVs in carcinogenesis, it has become increasingly clear that high-risk HPV E6 proteins have additional, p53-independent functions related to cellular immortalization. Analysis of E6 mutants suggests that at least some of these activities are dependent on E6AP. We therefore hypothesize that the high-risk HPVE6/E6AP complex recognizes and ubiquitinates a set of cellular proteins, which includes but is not limited to p53, and that targeting of these cellular proteins is linked to HPV-associated carcinogenesis. The first goal of this proposal is to characterize a set of proteins that we have shown to be targeted by the E6/E6AP complex and to determine if degradation of these proteins is relevant to cellular immortalization. These proteins are Scribble, Discs large (DIg), and utrophin. Interestingly, all three of these proteins are linked to architecture of multiprotein complexes formed at the plasma membrane, and work on Scribble and Dlg indicates that these are cooperating neoplastic tumor suppressors in Drosophila. While study of high-risk HPV E6 proteins led to discovery of E6AP and an established model for HECT E3 function, our understanding of the structure-function relationships of the E6 proteins, themselves, remains very limited. Several lines of evidence suggest that this is largely due to the difficulty of expressing properly folded E6 proteins for biochemical analyses. Recent advances in this area form the basis for the second goal of this proposal, characterization of the structure-function relationships of the HPV E6 proteins with respect to their role in facilitating protein ubiquitination. The third goal of this proposal is to further characterize specific aspects of the enzymatic mechanism of E6AP and HECT ubiquitin ligases, in general. Several of the approximately 30 human HECT E3s play critical roles in disease states in addition to cervical cancer, including Liddle's syndrome, Angelman's syndrome, and the life cycle of several types of viruses. A more thorough understanding of HECT E3 mechanism will therefore contribute to our understanding of several important health-related problems.